Drosophila pp 177-187 | Cite as

deGradFP: A System to Knockdown GFP-Tagged Proteins

  • Emmanuel Caussinus
  • Markus Affolter
Part of the Methods in Molecular Biology book series (MIMB, volume 1478)


Protein depletion by genetic means, in a very general sense including the use of RNA interference [1, 2] or CRISPR/Cas9-based methods, represents a central paradigm of modern biology to study protein functions in vivo. However, acting upstream the proteic level is a limiting factor if the turnover of the target protein is slow or the existing pool of the target protein is important (for instance, in insect embryos, as a consequence of a strong maternal contribution). In order to circumvent these problems, we developed deGradFP [3, 4]. deGradFP harnesses the ubiquitin-proteasome pathway to achieve direct depletion of GFP-tagged proteins. deGradFP is in essence a universal method because it relies on an evolutionarily conserved machinery for protein catabolism in eukaryotic cells; see refs. 5, 6 for review. deGradFP is particularly convenient in Drosophila melanogaster where it is implemented by a genetically encoded effector expressed under the control of the Gal4 system. deGradFP is a ready-to-use solution to perform knockdowns at the protein level if a fly line carrying a functional GFP-tagged version of the gene of interest is available. Many such lines have already been generated by the Drosophila community through different technologies allowing to make genomic rescue constructs or direct GFP knockins: protein-trap stock collections [7, 8] (,, P[acman] system [9], MiMIC lines [10, 11], and CRISPR/Cas9-driven homologous recombination.

Two essential controls of a protein knockdown experiment are easily achieved using deGradFP. First, the removal of the target protein can be assessed by monitoring the disappearance of the GFP tag by fluorescence microscopy in parallel to the documentation of the phenotype of the protein knockdown (see Note 1 ). Second, the potential nonspecific effects of deGradFP can be assessed in control fly lacking a GFP-tagged target protein. So far, no nonspecific effects of the deGradFP effector have been reported [3].

Key words

GFP Nanobodies F-Box Proteasome Gene expression 



The authors thank the Bloomington Drosophila Stock Center (Indiana University, Bloomington) for providing fly stocks, and Addgene for providing plasmids.


  1. 1.
    Dietzl G, Doris CD, Schnorrer F et al (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156CrossRefPubMedGoogle Scholar
  2. 2.
    Ni JQ, Zhou R, Czech B et al (2011) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8:405–407CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Caussinus E, Kanca O, Affolter M (2012) Fluorescent fusion protein knock-out mediated by anti-GFP nanobody. Nat Struct Mol Biol 19:117–121CrossRefGoogle Scholar
  4. 4.
    Caussinus E, Kanca O, Affolter M (2013) Protein knockouts in living eukaryotes using deGradFP and green fluorescent protein fusion targets. Curr Protoc Protein Sci 73:Unit 30.2Google Scholar
  5. 5.
    Ciechanover A (1998) The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J 17:7151–7160CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Varshavsky A (2012) The ubiquitin system, an immense realm. Annu Rev Biochem 81:167–176CrossRefPubMedGoogle Scholar
  7. 7.
    Morin X, Daneman R, Zavortink M et al (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A 98:15050–15055CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lowe N, Rees JS, Roote J et al (2014) Analysis of the expression patterns, subcellular localisations and interaction partners of drosophila proteins using a pigp protein trap library. Development 141:3994–4005CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Venken KJT, Carlson JW, Schulze KL et al (2009) Versatile P[acman] bac libraries for transgenesis studies in Drosophila melanogaster. Nat Methods 6:431–434CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Nagarkar-Jaiswal S, Lee PT, Campbell ME et al (2015) A library of Mimics allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila. Elife. doi: 10.7554/eLife.05338 Google Scholar
  11. 11.
    Nagarkar-Jaiswal S, DeLuca SZ, Lee PT et al (2015) A genetic toolkit for tagging intronic mimic containing genes. Elife. doi: 10.7554/eLife.08469 Google Scholar
  12. 12.
    Ciechanover A, Ben-Saadon R (2004) N-terminal ubiquitination: more protein substrates join in. Trends Cell Biol 14:103–106CrossRefPubMedGoogle Scholar
  13. 13.
    Jiang J, Struhl G (1998) Regulation of the hedgehog and wingless signalling pathways by the F-box/WD40-repeat protein Slimb. Nature 391:493–496CrossRefPubMedGoogle Scholar
  14. 14.
    Saerens D, Pellis M, Loris R et al (2005) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 352:597–607CrossRefPubMedGoogle Scholar
  15. 15.
    Rothbauer U, Zolghadr K, Tillib S et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889CrossRefPubMedGoogle Scholar
  16. 16.
    Tabata T, Eaton S, Kornberg TB (1992) The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev 6:2635–2645CrossRefPubMedGoogle Scholar
  17. 17.
    Le T, Liang Z, Patel H et al (2006) A new family of Drosophila balancer chromosomes with a w-Dfd-GMR yellow fluorescent protein marker. Genetics 174:2255–2257CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Pina C, Pignoni F (2012) Tubby-RFP balancers for developmental analysis: FM7c 2xTb-RFP, Cyo 2xTb-RFP, and TM3 2xTb-RFP. Genesis 50:119–123CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Matsumoto K, Toh-e A, Oshima Y (1978) Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4. J Bacteriol 134:446–457PubMedPubMedCentralGoogle Scholar
  20. 20.
    McGuire SE, Le PT, Osborn AJ et al (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302:1765–1768CrossRefPubMedGoogle Scholar
  21. 21.
    Tilmann B, Dominique F, Stefan L (2014) The transmembrane protein Macroglobulin complement-related is essential for septate junction formation and epithelial barrier function in Drosophila. Development 141:899–908CrossRefGoogle Scholar
  22. 22.
    Royou A, Field C, Sisson JC et al (2004) Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol Biol Cell 15:838–850CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zecca M, Struhl G (2007) Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation. Development 134:3001–3010CrossRefPubMedGoogle Scholar
  24. 24.
    Bopp D, Bell LR, Cline TW et al (1991) Developmental distribution of female-specific sex-lethal proteins in Drosophila melanogaster. Genes Dev 5:403–415CrossRefPubMedGoogle Scholar
  25. 25.
    Urban E, Nagarkar-Jaiswal S, Lehner CF et al (2014) The cohesin subunit Rad21 is required for synaptonemal complex maintenance, but not sister chromatid cohesion, during Drosophila female meiosis. PLOS Genetics 10:e1004540Google Scholar
  26. 26.
    Rubliaychaudhuri N, Dubruille R, Orsi GA et al (2012) Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm. PLoS Biol 10:e1001434CrossRefGoogle Scholar
  27. 27.
    Harder B, Schomburg A, Pflanz R et al (2008) Tev protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms. Biotechniques 44:765–772CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute of Molecular Life SciencesUniversity of ZurichZurichSwitzerland
  2. 2.Growth & Development, BiozentrumUniversity of BaselBaselSwitzerland

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